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Submitted on August 10, 2004
Accepted on October 12, 2004
III. Medical Department, University of Leipzig, Ph.-Rosenthal-Str. 27, D-04103 Leipzig, Germany; Institute for Molecular Pharmacology, Robert Roessle-Str. 10, D-13125 Berlin, Germany
* To whom correspondence should be addressed. E-mail: Ralf.Paschke{at}medizin.uni-leipzig.
The TSH receptor (TSHR) activates mainly two signal transduction pathways, cAMP production and phosphoinositides turnover, mediated by Gs- and Gq-coupling, respectively. Several activating deletion and point mutations within intracellular loop (ICL) 3 and the adjacent portion of transmembrane domain (TM) 6 support a direct G protein activation by this receptor domain. The ICL3, however, is predicted by modeling to interact with other receptor domains, primarily ICL2, to form a pocket for G protein binding and to allow optimum interaction. Systematic mutagenesis was used to identify important sites within ICL2 and potential interactions between ICLs 2 and 3 of the TSHR required for G protein coupling. Deletions of four to five residues and their corresponding multiple alanine substitutions were introduced into ICL2. Residues I523-D530, comprising mainly the N terminal half of ICL2, appeared to be critical for Gs- and Gq-mediated signaling. A single alanine substitution screening within ICL2 revealed hydrophobic residue M527 in particular and to lesser extents F525, R528, L529 and D530 as residues that selectively abolished or strongly impaired Gq-activation. Molecular modeling suggests that F525 interacts with ICL3. To test this hypothesis, ICL2/ICL3 double mutants introducing strong complementary properties were constructed and tested for functional rescue of Gq-mediated signaling. Our results indicate that ICL2 interacts with ICL3 in close vicinity to F525 and T607, suggesting a conformational cooperation between ICLs 2 and 3 during Gq-activation by TSHR.
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